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. Author manuscript; available in PMC: 2013 Apr 19.
Published in final edited form as: Vaccine. 2012 Mar 8;30(19):2937–2942. doi: 10.1016/j.vaccine.2012.02.072

Bioencapsulation of the hepatitis B surface antigen and its use as an effective oral immunogen

Celine A Hayden 1, Stephen J Streatfield 2,*, Barry J Lamphear 2, Gina M Fake 1, Todd K Keener 1, John H Walker 3, John D Clements 4, Debra D Turner 5, Ian R Tizard 5, John A Howard 1
PMCID: PMC3322283  NIHMSID: NIHMS366227  PMID: 22406456

Abstract

Hepatitis B remains a major global health problem despite the availability of a safe and effective vaccine. Segments of the population lack access to or respond poorly to the parenteral vaccine, perpetuating the infection-transmission cycle. A low cost, orally-delivered vaccine has the potential to alleviate many of these problems. Here we describe the expression of a bioencapsulated hepatitis B surface antigen (HBsAg) in maize and its immunogenicity, demonstrating for the first time a commercially feasible oral subunit vaccine production system for a major disease. This work surmounts previous barriers to plant-produced vaccines by expressing HBsAg at much higher levels and retaining antigen immunogenicity post-processing: factors which facilitated a robust immune response in mice without the need for an adjuvant. This method provides a practical solution to the delivery of a low-cost, stable oral vaccine.

Keywords: Hepatitis B, mucosal, oral vaccine, plant vaccine, bioencapsulation, immunogenicity, HBsAg

Introduction

Chronic infection with the hepatitis B virus (HBV) is a global problem despite the availability of a highly efficacious commercial vaccine. There are an estimated 240 million chronically infected patients worldwide who harbor and transmit HBV [1], 15 to 25% of whom will develop cirrhosis or cancer of the liver [2]. The virus is very stable, remaining viable on environmental surfaces ≥ 7 days, and can be transmitted by household (non-sexual) contact [2], highlighting the need for a low-cost, accessible vaccine.

Efforts to increase the availability of the injected HBV vaccine have significantly improved neonate vaccination rates worldwide (WHO, 2010), but there remain segments of the population that are poor responders. Dialysis patients, HIV-positive individuals, inflammatory bowel disease (IBD) patients, those with celiac disease, the morbidly obese, and the elderly are all at risk of low antibody titer (<10mIU/mL) after receiving the standard three injected doses [3–10] These studies have demonstrated that seroconversion in these groups ranges from 64% in dialysis patients to less than 30% in the elderly. There are also relatively low compliance rates among adults at high risk of contracting HBV, such as healthcare workers, sex workers, men who have sex with men, and intravenous drug users [11–16].

The elimination of chronic hepatitis B infections is an achievable goal [17, 18]. Development of an oral vaccine could facilitate eradication by providing a more convenient and cost-effective means of vaccinating individuals, eliminating the need for needles and trained medical staff for administration, and infrastructure for waste disposal. Oral vaccines may also induce a more robust immune response in poor responders by activating antigen presenting cells (APCs) in the mucosa of the oral cavity [19, 20], as well as APCs in the Peyer’s patches of the gut [21].

Presently, commercialized vaccines for hepatitis B consist of a recombinant surface antigen (HBsAg) produced in yeast and purified prior to intramuscular administration. Plant-based vaccines offer the potential for an orally-delivered vaccine without the need for purification, thereby eliminating most of the expense normally associated with vaccines. There have been several attempts to express HBsAg in various edible plant systems for oral delivery [22–24], but none of these systems offer a practical vaccine candidate. Major drawbacks to these systems include their relatively low levels of HBsAg expression and the inability to process the material to an edible form without the need for extraction and purification. In the best reported case to date, a clinical trial with potato-expressed HBsAg induced increases in anti-HBsAg titers in up to 63% of volunteers [25]. While this is encouraging, an increase in HBsAg exposure was needed for a more effective immunologic response. The consumption of potato is limited by the palatability and indigestibility of raw potato starch. Unfortunately, processing the material to render it more palatable drastically reduced its immunogenicity [26].

Using maize as an oral vaccine delivery system circumvents digestibility and palatability issues encountered in other edible plant systems. Maize does not contain highly allergenic, anti-nutritional, or carcinogenic, agents and is therefore a safe vehicle for administration. It also provides a bioencapsulated environment that is rich in protease inhibitors, low in moisture content, and high in carbohydrates, conditions conducive to antigen stability under ambient conditions as well as those encountered in the gastrointestinal tract [27, 28].

Previous studies with other antigens have demonstrated the protective [29] and immunostimulatory effect of maize-expressed vaccine candidates [30–32]. Here we describe the conditions necessary for obtaining high levels of HBsAg expression in grain, enabling a strong immune response in mice against HBsAg. The results described below warrant a renewed interest in plant-based oral vaccines for a more convenient, cost-effective, and efficacious product.

Materials and methods

Construct design

All construct contain the small form of HBsAg (Genbank accession S62754.1), codon optimized for maize and built from oligonucleotides in the manner of Stemmer et al .[33]. The localization of proteins by specific targeting sequences is known and was used to direct the HBsAg to specific intracellular locations [34]. Construct HBA used a maize polyubiquitin promoter for expression of HBsAg targeted to the cell wall using a codon optimized type B barley alpha amylase signal sequence (BAASS) [35] at the N-terminus. BAASS has been shown to target maize-expressed proteins to the cell wall [36], HBC used the polyubiquitin promoter and the amyloplast signal sequence (MAALATSQLVATRAGLGVPDASTFRRGAAQGLRGARASAAADTLSMRTSARA APRHQQQARRGGRFPSLVVC) to target to the vacuole.. HBD was constructed as in HBA with an ER-targeting signal (KDEL) at the C-terminus. HBE contained the 1.4kb globulin1 promoter (NCBI Gene ID 732801) and expressed a BAASS:HBsAg fusion protein. All constructs contained a potato protease inhibitor II (Pin II) termination sequence [37] and the glyphosate resistance gene [38].

Immunoblot

Protein was obtained by three consecutive extractions from 100mg of ground maize material, the first two extractions in 1mL of PBS+0.05%Tween, and a final extraction in 1mL PBS+1%TritonX-100. Extracts and yeast-derived rHBsAg (Meridian Life Sciences #R86872) were subjected to non-reducing (0mM DTT) or reducing (50mM DTT) conditions, heated to 70°C for 10 minutes, loaded onto a 10% Bis-Tris SDS PAGE gel, and transferred to a PVDF membrane using the iBlot system (Invitrogen). Rabbit anti-HBsAg (Genway#18-511-245179), AP-conjugated goat anti-rabbit IgG (Jackson#111-055-003), and BCIP/NBT liquid substrate (Sigma#B1911) were used to visualize HBsAg bands..

Maize transformation and seed propagation

Constructs were transferred into Agrobacterium tumefaciens and maize as described previously [39]. Transformation events were selected using bialaphos and propagated as described previously [38, 40]. Lines showing highest HBsAg expression were chosen for backcrosses into elite inbred lines SP122 and SP114. Homozygous lines were obtained by selfing plants twice and screening for correct segregation ratios. Homozygosity was confirmed by assaying 20 single seeds for HBsAg (see below). Homozygous lines were crossed (SP122-HBsAg × SP114-HBsAg) to produce hybrid seed. These were grown and self-pollinated to produce hybrid grain. To obtain additional grain for the mouse trial, HBE was backcrossed six times into the SP114 genetic background and selfed once.

Antigen detection

Protein extractions were performed on either a single ground seed, or 100mg of ground bulk seed, in 1mL of extraction buffer (PBS+1%TritonX-100). Six T1 seeds were assayed from each ear while 50-seed bulks of subsequent generations were sampled in duplicate. Total soluble protein in the supernatant was determined using a Coomassie Brilliant Blue assay [41]. HBsAg was detected by sandwich ELISA in which rabbit anti-HBsAg was used as coating antibody (GenWay, cat# 18-511-245179) and biotinylated anti-HBsAg (Meridian Life Science, cat# B65811B) was used as secondary antibody. Recombinant HBsAg (GenWay, cat# 10-663-45361) was used to generate a standard curve. Estimates of HBsAg absorption and degradation through the GI tract assume 15g germ were ingested over 3 days and condensed into 1g of fecal material over 3 days. Pellets were sampled on the third day of the first oral boost.

Seed processing

HBE lines were soaked in water to approximately 50% moisture and germ was extracted by hand, dried overnight at 37°C (6–15% moisture), and ground. Oil was extracted using 5mL hexane per gram of germ. The residual hexane was evaporated in a hood.

Mouse study

Eight BALB/c mice for each treatment were vaccinated with an intraperitoneal injection of 0.5μg Recombivax® (commercial HBsAg vaccine) on day 0 and boosted 13, 15, and 17 weeks post-injection. Mice were fasted the night before oral boosting to improve ingestion of germ material on day 1 of boosting. Immediately prior to feeding, 5g of germ was formed into a pellet with 5mL of ddH2O or ddH2O+25μg LT(R192G/L211A) and placed in individual cages for consumption. Each boost consisted of three consecutive daily doses of defatted germ.

Anti-HBsAg antibody detection

Blood samples were collected by submandibular venous puncture every 2 or 3 weeks (see Figure 5), centrifuged to remove red blood cells, and stored in 50% glycerol at −80°C. Fecal material was obtained from cages cleaned 24 hours prior to collection and stored at −80°C. Fecal sampling occurred the morning of injection (day 0) and twice a week once oral boosting was initiated. Serum anti-HBsAg IgG and IgA were detected using a sandwich ELISA. Plates were coated with rHBsAg (Meridian, cat# R86872), serum samples diluted 1:250, and AP-conjugated anti-mouse IgG (Jackson Immunoresearch Laboratories, cat# 115-055-008) or anti-mouse IgA (Abcam, cat# ab972) used to detect IgG and IgA, respectively. For secretory IgA, 100mg of fecal pellets were resuspended in 1mL protease inhibitor cocktail solution (Roche, cat#11836153001), diluted an additional 1:100 and used in place of serum samples in the aforementioned sandwich ELISA. Buffer alone produced a mean O.D.405 of 0.12, which was subtracted from all readings.

Figure 5.

Figure 5

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Serum anti-HBsAg a) IgG and b) IgA response as determined by a sandwich ELISA.. Normalized O.D. values were calculated for each mouse relative to its own value just prior to oral boost (O.D. at weekn/O.D. at week12), and geometric means of normalized values were calculated for each group of 8 mice. White arrows represent primary injection of Recombivax and black arrows indicate oral boosts.

A direct enzyme immunoassay (Diasorin, cat# P001931) quantified serum anti-HBsAg antibodies in mIU/mL using the WHO First International Standard. Samples were diluted within the linear range of the assay and used according to kit specifications.

Statistical analysis

Each of the four boosting treatments comprised eight mice. Fecal titers were plotted using arithmetic means, and differences in fecal IgA responses between adjuvanted and non-adjuvanted HBsAg germ were tested using the non-parametric Mood’s Median Test for samples collected 130 days post-injection. Serum IgG and IgA O.D. values were normalized to values detected at bleed 6 (bleedn O.D./bleed6 O.D.) and a logarithmic transformation of the data was effectuated before ANOVA analysis of the terminal bleed (bleed 9) over all treatments. Geometric means were calculated and plotted to reflect the logarithmic transformation of the data. Changes in anti-HB titers (mIU/mL) between bleed 6 (pre-boost) and bleed 9 (post-boost) were based on arithmetic means and analyzed using non-transformed data in an ANOVA. To determine whether changes were significantly different from zero, a Bonferroni-adjusted 99% confidence interval was used for all four treatments so that the overall confidence in all estimates was greater than 95%.

Results

HBsAg expression in maize

To optimize production of HBsAg in maize, constructs containing different promoters and targeting signals were assembled and transformed into maize (Figure 1). Either the constitutive polyubiquitin or the embryo-preferred globulin1 promoter was used to drive expression of HBsAg, and various signal sequences were tested for their effect on HBsAg accumulation in grain [42, 43].

Figure 1.

Figure 1

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Construct design for expression of HBsAg in Zea mays. UBI, Ubiquitin promoter; Glob1, globulin1 promoter; BAASS, barley alpha amylase signal sequence; KDEL, ER targeting sequence; PinII, potato proteinase inhibitor II termination sequence. All constructs also contained an herbicide resistance gene following the PinII termination sequence.

Constructs were transformed into Agrobacterium and subsequently into maize. Single seeds from the initial transformant (T1 seed) were analyzed by a sandwich ELISA for HBsAg expression (Figure 2). Approximately equivalent levels of HBsAg were produced whether the antigen was targeted to the cell wall (construct HBA) or ER (construct HBD) when driven by the polyubiquitin promoter. Recombinant protein directed to the vacuole (construct HBC) only resulted in a few transformants and the plants died before maturation. While the targeting sequences influence the expression the specific intracellular locations HBsAg were not confirmed. Expression levels were highest when HBsAg was expressed by the embryo-preferred promoter, globulin1 (construct HBE). The highest single seed for this construct produced over 0.25% total soluble protein (TSP), more than two fold greater antigen than the level produced by the polyubiquitin promoter. These data demonstrate the utility of using an embryo-specific promoter for the high level production of recombinant proteins.

Figure 2.

Figure 2

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Expression from single seed in the first generation after transformation (T1), as determined by a sandwich ELISA. Each bar represents the highest expressing seed from a given ear. The top ten expressing ears are displayed for each construct. Expression levels are reported as % total soluble protein (%TSP).

The presence of the HBsAg protein was confirmed by immunoblot (Supplementary Figure 1 online). Furthermore, there is evidence that disulfide linkages are important for HBsAg antigen presentation and formation of virus-like particles [44], therefore maize extracts were subjected to non-reducing as well as reducing conditions to detect antigen multimers. Under reducing conditions, monomers of HBsAg are most prevalent, while under non-reducing conditions the preferred conformation is dimerization (Supplementary Figure 1 online).

To further enhance HBsAg accumulation, seed was selected from two high-expressing and high-yielding HBE ears to initiate a backcrossing program. The average HBsAg level of positive seeds from these selected ears was 0.08% total soluble protein (TSP). These plants were backcrossed to elite inbred SP122 and SP114 lines and were then selfed twice to make homozygous plants which were identified by herbicide screening. Homozygous parents were then crossed to generate hybrid seed which in turn was grown and selfed to produce hybrid grain. The level of HBsAg for each of these lines was determined, with the hybrid grain exhibiting the highest level, 0.46%TSP (71μg/g) (Figure 3).

Figure 3.

Figure 3

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Increases in HBsAg %TSP gained from the production of hybrid grain. Expression is reported as the average of all positive single seeds from the ear chosen for subsequent breeding (T1 seed) or as the highest expressing ear (50-seed bulk) from a given generation. T1 seed was used to generate homozygous (hmz) lines in the SP122 and SP114 genetic backgrounds, and these homozygotes were used to produce hybrid seed (from SP122×SP114) and hybrid grain (from selfed hybrid plants).

Bulking of all ears from all hybrid grain resulted in maize material containing 0.20%TSP (29μg/g) that was used for subsequent mouse feeding studies. Additional grain for mouse trials was obtained by backcrossing T1 seed six times to an elite inbred and selfing once (see Methods). This backcross bulk resulted in HBsAg expression of 0.15%TSP (27μg/g). Both bulked hybrid and backcross seed batches were combined and used for further processing of the HBE seed.

The HBE construct contains the globulin1 embryo (germ) preferred promoter, therefore separation of the germ from the endosperm can increase recombinant protein concentrations significantly, as has been shown for other recombinant proteins [30, 45]. Following germ enrichment and oil extraction, maize material exhibited an increase in HBsAg concentration. While the %TSP remained equivalent to the pre-processed maize material (0.15%), the concentration of HBsAg per dry weight increased 6-fold to 166μg HBsAg/g maize material, effectively increasing the amount of HBsAg administered per dose in the subsequent mouse trial (see below).

Oral administration of HBsAg

To determine whether germ-enriched HBE maize material could stimulate a robust humoral response in mice, BALB/c mice were given a primary injection with the commercial vaccine followed by an oral boosting regime with HBsAg or control (non-transgenic) germ. Serum IgG was monitored every 2 weeks during the primary response and boosts were administered once the primary response peaked. As anticipated, all mice elicited an initial response to the injected vaccine, which peaked at approximately 10 weeks post-injection. Mice were fed 5g of either defatted control or defatted HBsAg germ (a total of 0.83mg HBsAg) on three consecutive days, resulting in exposure to 2.5mg HBsAg per boost. For each treatment, eight mice were used. A total of three oral boosts were administered two weeks apart (weeks 13, 15, and 17) for each of the different groups of mice. Evidence of HBsAg absorption and degradation was apparent in fecal material where < 2% of the ingested HBsAg was detectable by a sandwich ELISA (data not shown). This degradation of maize-derived HBsAg through the GI tract is consistent with other maize-derived recombinant proteins [29]. To monitor mucosal production of anti-HBsAg secretory IgA, fecal material was assayed. Mice fed control germ with or without LT(R192G/L211A) adjuvant showed no change in anti-HBsAg IgA (Figure 4) over the course of the experiment. In contrast, all mice receiving the HBsAg germ with or without the LT adjuvant showed a greater than 50% increase in anti-HBsAg IgA O.D. values. There was no significant difference between LT-adjuvanted HBsAg germ and non-adjuvanted HBsAg germ (p-value=0.189). Additionally, titers fell after two weeks but increased over previous levels with each oral boost. These IgA titers in fecal material clearly demonstrate induction of the immune response at mucosal sites as a result of oral administration of HBsAg germ and illustrate the potential effectiveness of orally administered subunit vaccines.

Figure 4.

Figure 4

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Fecal anti-HBsAg sIgA levels in mice as determined by a sandwich ELISA. Mean O.D. values were determined for each treatment of 8 mice. Black arrows indicate the initiation of oral boosting.

To determine the systemic immunologic effect of the oral antigen, serum anti-HBsAg IgG and IgA were monitored at two-week intervals. Mice developed a primary antibody response to the Recombivax injection that peaked at approximately week 10 (Figure 5). Results were normalized to antibody titers at week 12 for each individual mouse in order to compare pre- and post-boost responses more accurately among treatments. Mice fed control germ with or without LT showed a decrease in mean titer after initiation of oral boosting (Figure 5). In contrast, mice treated with HBsAg germ showed significant increases in both IgA and IgG after oral boosting (p-value<0.0001, and p=0.005, respectively). These trends did not differ significantly between the LT-treated and non LT-treated mice (p-value = 0.774 and 0.603, respectively). Anti-HBsAg IgA titers showed the strongest response, with HBsAg germ eliciting a greater than 50% increase in 6 out of 8 mice treated with or without adjuvant. The amplitude of the IgG response was smaller, but still increased more than 20% in 5 out of 8 mice for both HBsAg treatments.

In order to compare these antibody responses to previous studies in the literature, total anti-HBsAg titers in mIU/mL were assessed. Seroconversion in humans using this standard is considered complete with titers greater than 10mIU/mL [46]. Prior to oral boosting, the mice reached a mean titer of 3488mIU/mL, indicating that the primary dose via intraperitoneal injection was more robust than expected. Serum samples were compared from week12 (pre-boost) and week19 (post-boost, terminal bleed). Mice did not show a statistically significant change in titer when fed control germ alone (99% CI, −1036 to 2226) or with the LT adjuvant (99% CI, −2390 to 873; Figure 6) while mice fed the HBsAg germ without adjuvant showed a mean increase in titer of 3003 mIU/mL after oral boosting. The change in antibody titer appeared to be different between LT adjuvanted and non-adjuvanted HBsAg germ (Figure 6), but the change was not statistically significant (p=0.908). Seven out of eight mice in these two treatments showed greater than 50% increases in total antibody titer. Interestingly, one of the eight mice fed control germ and three of the eight mice fed control germ with LT showed > 50% increases.

Figure 6.

Figure 6

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Total change in serum anti-HBsAg Ig in mice fed control versus HBE germ with or without the LT(R192G/L211A) adjuvant. Values were determined using serum Ig levels in week12 (pre-boost) and week19 (post-boost).

Discussion

Maize-produced vaccine candidates have shown promise for several model diseases and this approach was adopted for hepatitis B. Using a construct with an embryo-preferred promoter, plants were transformed and high expressing HBsAg lines were identified. These were then bred into elite corn backgrounds and selected at each generation to further increase the concentration of antigen by 2.5-fold. Finally, physical processing of the seed concentrated the antigen another 6-fold resulting in material with a 20-fold greater concentration than the best expression previously reported in plants [23]. This allowed for higher doses to be easily administered to animals with the goal of increasing the immune response. Additionally, dimer formation of HBsAg was confirmed by immunoblot (Supplementary Figure 1, online) suggesting that the maize-produced HBsAg correctly presents antigenic sites and is a good candidate for an oral-based vaccine.

When mice were fed the HBsAg germ as an oral boost, mucosal responses as measured by anti-HBsAg IgA in fecal matter were dramatic, undergoing amplification with each oral boost. The amplification of antibody levels at subsequent boosts may indicate an establishment of immunological memory at mucosal sites but this observation will need to be further validated in longer-term studies.

Previous efforts to induce an immune response using HBsAg in potato with an adjuvant elicited increases of 1000mIU/mL in mouse serum. In contrast, when maize HBsAg material was fed under similar conditions, mice elicited a greater than 3000mIU/mL increase, even in the absence of an adjuvant. Furthermore, the amplitude of the response to the maize booster may have been suboptimal since the initial antibody response elicited by the primary injection was already high. Late immunologic responses could account for some of these high titers since some of the mice fed control germ exhibited increasing serum titers at the time of oral boosting yet showed no indication of eliciting a response in fecal material. Immunologic responses may conceivably have been more divergent between germ treatments had the primary response been less robust. The strong increase observed in spite of high pre-boost antibody levels suggests a promising outcome for this material’s use in human volunteers.

In human clinical trials, the potato material cited above stimulated humoral responses in 53–63% of adult volunteers, depending on the dosing regimen. While very encouraging, this response rate is lower than the 85–100% of individuals that respond to the commercial parenteral vaccine [47]. Unfortunately, practical problems of digestibility and palatability of raw potatoes limited the viability of administering higher doses of antigen. In theory, boiling the potato tissue could produce a more palatable product which could result in ingestion of higher doses, but in practice antigen efficacy is reduced by 25-fold after boiling [26].

In contrast, the outlook for maize is much more promising. The maize material can deliver a much larger dose in a smaller amount of material and corn does not suffer from the digestibility problems associated with potatoes. The maize-produced HBsAg elicited a strong IgA response that is important for pathogens that enter through the mucosal route. Induction of immunologic responses at the mucosa may be a mechanism to induce protection in parenteral non-responders. Evidence of enhanced protection from maize-based oral vaccines relative to parenteral vaccines has been demonstrated in pigs challenged with transmissible gastroenteritis virus (TGEV; [30]).

Maize seed can be processed to deliver precise antigen doses in easily palatable material that is stable at above-ambient temperatures, indicating another practical aspect of this approach. This temperature stability could enable transportation of oral vaccines to remote locations without the need for a cold chain. Furthermore, the constructs and maize lines used in this study were non-optimized in terms of expression, and additional gains in antigen concentration are realizable in the near future. Extending this work to clinical trials will provide the next step towards a realistic approach to the oral administration of subunit vaccines.

In addition to performing human clinical trials there are several other important factors that require verification. Long-term studies are necessary to establish the storability of the processed corn material at ambient temperatures and to assess immunological memory establishment in animals after receiving an oral dose. To effectuate the greatest impact will also require the use of a maize oral vaccine as a primary vaccine dose as well as a booster. This would not only reduce vaccine costs in developed countries but also allow more widespread administration of the vaccine worldwide and in effect help decrease the disease burden on a global scale.

Supplementary Material

01

Supplementary Figure 1. HBsAg monomer and dimer formation as resolved by SDS-PAGE immunoblot under non-reducing (A) and reducing (B) conditions. A) Lane1:,control (non-transgenic) maize extract, Lane 2:,Invitrogen pre-stained protein ladder, Lane 3: 100ng yeast-derived recombinant HBsAg, Lane 4: HBsAg maize extract. B) Lane1: 100ng yeast-derived recombinant HBsAg, Lane2: HBsAg maize extract. Single and double asterisks indicate monomeric and dimeric forms of HBsAg, respectively. HBsAg-specific bands appear at approximately 25kDa and 50kDa in the yeast and maize-derived extracts but not in the control maize extract. The yeast-derived HBsAg under non-reducing conditions shows a faint band at 50kDa and a smear at high molecular weights, indicating aggregation of the antigen. Ladder bands represent 181.8, 115.5, 82.2, 64.2 (red band), 48.8, 37.1, 25.9, 19.4, 14.8, 6.0kDa. All maize extract lanes contain 3μg of total soluble protein, as determined by Bradford assay.

Highlights.

  • Hepatitis antigens expressed in maize can provide a commercially-feasible oral vaccine

  • Elevated levels of HBsAg were achieved in maize seed with embryo-preferred promoters.

  • Optimization of genetic background and processing resulted in high HBsAg levels

  • Mice displayed robust systemic and mucosal immune responses after oral boosting

  • Immunogenicity of the material was not adjuvant-dependent

Acknowledgments

This project was supported by NIH grants 1R43AI068239-01A1 and 3R43 AI068239-01A1S1

Footnotes

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Associated Data

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Supplementary Materials

01

Supplementary Figure 1. HBsAg monomer and dimer formation as resolved by SDS-PAGE immunoblot under non-reducing (A) and reducing (B) conditions. A) Lane1:,control (non-transgenic) maize extract, Lane 2:,Invitrogen pre-stained protein ladder, Lane 3: 100ng yeast-derived recombinant HBsAg, Lane 4: HBsAg maize extract. B) Lane1: 100ng yeast-derived recombinant HBsAg, Lane2: HBsAg maize extract. Single and double asterisks indicate monomeric and dimeric forms of HBsAg, respectively. HBsAg-specific bands appear at approximately 25kDa and 50kDa in the yeast and maize-derived extracts but not in the control maize extract. The yeast-derived HBsAg under non-reducing conditions shows a faint band at 50kDa and a smear at high molecular weights, indicating aggregation of the antigen. Ladder bands represent 181.8, 115.5, 82.2, 64.2 (red band), 48.8, 37.1, 25.9, 19.4, 14.8, 6.0kDa. All maize extract lanes contain 3μg of total soluble protein, as determined by Bradford assay.

NIHMS366227-supplement-01.tif (878KB, tif)

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